Systems and methods for electrophoretic separation and analysis of analytes

ABSTRACT

A system for assaying a biological sample for a presence of a target analyte includes an assaying device and a computer controller. The assaying device includes a housing, a receptacle disposed in the housing, and a source of activation energy. The receptacle is configured to accept an electrophoresis cell. The electrophoresis cell has a recess area configured to accept a chip configured to accept the biological sample. The chip includes a polymeric separation medium with activatable functional groups that covalently bond to the target analyte when activated. The source of activation energy is configured to supply activation energy to activate the activatable functional groups. The computer controller is operably coupled to the source of activation energy and is configured to activate the source of activation energy to direct an application of activation energy to the polymeric separation medium to activate the activatable functional groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/160,636 entitled “System and Methods for Electrophoretic Separationand Analysis of Analytes,” filed May 20, 2016, which claims priority toand the benefit of U.S. Provisional Patent Application Ser. No.62/164,495 entitled, “Systems and Methods for Electrophoretic Separationand Analysis of Analytes,” filed May 20, 2015, the disclosure of each ofwhich is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Award numberR43GM112236 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The present disclosure provides devices, systems, and methods forassaying a biological sample for a presence of one or more targetanalytes, such as target proteins.

Understanding cell-to-cell variation in protein expression is a largepart of understanding the pathogenesis of tumors, characterizing thedifferentiation states of stem cells, and developing well-controlled andfunctionally-validated in vitro human ‘disease in a dish’ models fordrug development, target screening, and toxicity studies. Single-cellanalysis is of growing importance, but has been largely limited totranscriptome (e.g., RNA) and genome (e.g., DNA) technologies.Importantly, these measurements do not always correlate with the proteinlevels that dictate phenotype. Single-cell protein measurementsrepresent an unsurmounted hurdle in stem cell, cancer, and immunologyresearch.

Methods to determine protein level expression of a biological sampleinclude electrophoresis followed by probe analysis. Electrophoresis is atechnique that typically applies an electric field to a biologicalsample in a separation medium, such that individual molecules of thebiological sample will disperse throughout the separation medium basedon molecular size and charge. Cell lysis prior to electrophoresis, mayreduce the biological sample to a set of individual molecules that caneffectively separate throughout the separation medium. Cell lysis,electrophoresis, and subsequent probe analysis are typically executed onseparate platforms with extensive user manipulation.

Thus, a need exists for devices, systems, and methods for assaying for apresence of various types of analytes using a single, self-containedsystem.

SUMMARY

An aspect of the present disclosure provides a system for assaying abiological sample for a presence of one or more target analytes,comprising a housing comprising (i) a receptacle that is configured toaccept an electrophoresis cell having a recess area configured to accepta chip including a polymeric separation medium having activatablefunctional groups that covalently bond to the one or more targetanalytes when activated, wherein the chip accepts the biological sample;and (ii) a source of activation energy that supplies activation energyto activate the activatable functional groups. The system furthercomprises a computer controller operatively coupled to the source ofactivation energy and programmed to direct application of activationenergy from the source of activation energy to the polymeric separationmedium to activate the activatable functional groups.

In some embodiments, the electrophoresis cell is removable. In someembodiments, the chip is consumable. In some embodiments, the housinghas a length that is less than about 50 cm, a width that is less thanabout 50 cm, and/or a height that is less than about 50 cm. In someembodiments, the length is less than about 40 cm, the width is less thanabout 40 cm, and/or the height is less than about 40 cm.

In some embodiments, the polymeric separation medium includes aplurality of microwells formed therein. In some embodiments, themicrowells are dimensioned to accommodate a single cell from thebiological sample in an individual microwell of the plurality ofmicrowells, and wherein the individual microwell has a dimension of 100□m or less.

In some embodiments, the polymeric separation medium is cross-linked. Insome embodiments, the activatable functional groups are benzophenonegroups. In some embodiments, the activatable functional groups areactivatable by electromagnetic radiation. In some embodiments, theelectromagnetic radiation is visible light, ultraviolet (UV) light, orinfrared light.

In some embodiments, the computer controller is included in the housing.In some embodiments, the computer controller is included in anelectronic device that is remotely situated with respect to the housing.In some embodiments, the system further comprises a power source thatprovides power to the computer controller to assay the biological samplefor the presence of the one or more target analytes.

In some embodiments, the source of activation energy is a source ofultraviolet radiation. In some embodiments, an external face of thesource of activation energy comprises an optical filter. In someembodiments, the optical filter is configured to pass a subset ofwavelengths compatible with the assaying through the optical filter. Insome embodiments, the optical filter is configured to reducecondensation of buffer solution.

In some embodiments, the system further comprises an electronic displayoperatively coupled to the computer controller, wherein the electronicdisplay has a user interface that permits a user to instruct thecomputer controller to assay the biological sample.

In some embodiments, the electrophoresis cell comprises one or moreelectrodes on opposing sides of the recess area. In some embodiments,the system further comprises a power source that provides power to theone or more electrodes to generate an electric field across the recessarea. In some embodiments, the computer controller is programmed todirect field strength of the electric field across time. In someembodiments, the system further comprises a plurality of slots formed inthe housing configured to accommodate one or more pins, wherein anelectrical connection is formed when the one or more pins are adjacentto one or more conductive contact pads of the electrophoresis cell. Insome embodiments, the computer controller is programmed to directvoltage polarity of each of the one or more pins to control a directionof the electric field. In some embodiments, the one or more pins arespring-loaded pins. In some embodiments, the plurality of slots isconfigured to accommodate a plurality of electrophoresis cell formfactors and/or a plurality of electrode configurations. In someembodiments, the system further comprises one or more leveling sensorsthat permit a solution in the recess area to be leveled to reducespatial perturbations in the electric field. In some embodiments, thesystem further comprises one or more leveling indicators that provide anindication as to a degree of leveling of the solution in the recessarea.

In some embodiments, the system further comprises a sensor operativelycoupled to the computer controller, wherein the sensor permitsidentification of one or more identification members on the chipinserted into the receptacle. In some embodiments, the one or moreidentification members include one-dimensional or two-dimensionalidentification barcodes. In some embodiments, the one or moreidentification members include radio frequency identification (RFID)units. In some embodiments, each of the one or more identificationmembers is unique. In some embodiments, the system further comprisesmemory coupled to the computer controller, wherein the memory stores anidentification of the one or more identification members. In someembodiments, the computer controller is programmed to transmit orretrieve an identification of the one or more identification membersfrom memory of a remote computer system. In some embodiments, a firstidentification member of the one or more identification membersdetermines a first set of assay parameters for the assaying. In someembodiments, the computer controller is programmed to upload a givenidentification member and/or a set of assay parameters. In someembodiments, the sensor is an optical sensor that optically identifiesthe one or more identification members.

In some embodiments, the housing further comprises a source of solution.In some embodiments, the computer controller is programmed to directflow of a solution from the source of solution to the chip. In someembodiments, the system further comprises one or more valves operativelycoupled to a reservoir of the source of buffer solution, wherein thecomputer controller is programmed to open or close the one or morevalves to direct the flow of the solution from the reservoir to thechip. In some embodiments, the solution is a buffer solution. In someembodiments, the computer processor is programmed to separatelyintroduce at least a first solution and a second solution from thesource of solution to the chip. In some embodiments, the computerprocessor is programmed to separately introduce two or more solutionssequentially from the source of solution to the chip.

In some embodiments, the system further comprises one or moreinterlocking switches. In some embodiments, locking the one or moreinterlocking switches permits activation of the source of activationenergy. In some embodiments, locking the one or more interlockingswitches permits activation of a power source.

In some embodiments, the system further comprises one or more weirstructures formed in the electrophoresis cell. In some embodiments, theone or more weir structures trap bubbles on a top surface of the chip.In some embodiments, the one or more weir structures prevent bubblesfrom floating on a top surface of the chip. In some embodiments, the oneor more weir structures reduce perturbation in the electric field,reduce blockage of wavelengths generated by the source of activationenergy, dampen fluid motion of buffer solution, or combinations thereof.

In some embodiments, the system further comprises a rinsing fixture,wherein an angle of the rinsing fixture controls a flow rate of buffersolution to the chip. In some embodiments, the system further comprisesa probing fixture adjacent to the chip. In some embodiments, the systemfurther comprises one or more pouring or spout features formed in theelectrophoresis cell to aid in emptying the electrophoresis cell.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various respects,all without departing from the disclosure. Accordingly, the drawings anddescription are to be regarded as illustrative in nature, and not asrestrictive.

In some embodiments, a system for assaying a biological sample for apresence of a target analyte includes an assaying device and a computercontroller. The assaying device includes a housing, a receptacledisposed in the housing, and a source of activation energy. Thereceptacle is configured to accept an electrophoresis cell. Theelectrophoresis cell has a recess area configured to accept a chipconfigured to accept the biological sample. The chip includes apolymeric separation medium with activatable functional groups thatcovalently bond to the target analyte when activated. The source ofactivation energy is configured to supply activation energy to activatethe activatable functional groups. The computer controller is operablycoupled to the source of activation energy and is configured to activatethe source of activation energy to direct an application of activationenergy to the polymeric separation medium to activate the activatablefunctional groups.

In some embodiments, an apparatus includes a housing, a receptacle, aradiation energy source, an electric power source, and a computercontroller. The housing includes a base and a lid. The receptacle isdefined by an interior surface of the base and is configured to receivean electrophoresis cell. The electrophoresis cell is configured to becoupled to a chip including a polymeric separation medium withfunctional groups configured to covalently bond to a target analytewithin a biological sample disposed on the chip in response to beingactivated. The radiation energy source is coupled to an interior surfaceof the lid and is configured to supply activation energy operable toactivate the functional groups. The electric power source is disposedwithin the housing and fluidically isolated from the receptacle. Theelectric power source is configured to be electrically coupled to theelectrophoresis cell when the electrophoresis cell is disposed in thereceptacle. The computer controller is coupled to the housing andfluidically isolated from the receptacle. The computer controller isoperable to activate the radiation energy source and the electric energysource when the electrophoresis cell is disposed in the receptacle toassay the biological sample.

In some embodiments, an electrophoresis cell is configured to bedisposed within a receptacle of an assay device to assay a biologicalsample for a presence of a target analyte. The electrophoresis cellincludes a body, a chip, a conductive contact pad, an electrode, and aweir structure. The body defines a recess area configured to receive thechip such that a surface of the chip is substantially flush with asurface of the body. The chip including a polymeric separation mediumwith functional groups configured to covalently bond to a target analytewithin the biological sample disposed on the chip in response to beingactivated. The conductive contact pad is coupled to the body and isconfigured to be electrically connected to a power supply of the assaydevice when the electrophoresis cell is disposed within the receptacle.The electrode is disposed within the body and is electrically connectedto the conductive contact pad. The electrode is configured to produce anelectric field across the recess area in response to a flow of electriccurrent from the power supply when the electrophoresis cell is disposedwithin the receptacle. The weir structure is disposed within the body onat least one side of the recess area. The weir structure is configuredto control a flow of a solution across the recess area.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the present disclosure are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of the present disclosure will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the presentdisclosure are utilized, and the accompanying drawings (also “figure”and “FIG.” herein), of which:

FIG. 1 illustrates a single cell Western Blotting (scWB) array image andassay workflow schematic, according to an embodiment.

FIG. 2 illustrates a main unit of a scWB apparatus, according to anembodiment.

FIG. 3 illustrates a receptacle of the main unit 100 of FIG. 2 thatholds a modular electrophoresis cell which itself may contain aremovable chip.

FIG. 4 illustrates one embodiment of a modular removable electrophoresiscell configured for use in the main unit 100 of the scWB apparatus ofFIG. 2.

FIGS. 5A and 5B illustrate cross-sectional views of the modularremovable electrophoresis cell of FIG. 4, containing a removable chip.

FIGS. 6A-6C and 7A-7C illustrate cross-sectional views of the modularremovable electrophoresis cell of FIG. 4, in fluid communication with areservoir and valve features for addition of one or more buffersolutions into the reservoir of the modular removable electrophoresiscell of FIG. 4.

FIGS. 8A-8C illustrate a device for performing antibody probing of aremovable chip according to an embodiment.

FIG. 9 illustrates a cross-sectional view of an alternate constructionfor an antibody incubation fixture according to an embodiment.

FIG. 10 illustrates a rinsing fixture according to an embodiment.

FIG. 11 shows a computer control system that is programmed or otherwiseconfigured to implement any of the methods provided herein.

FIGS. 12A and 12B illustrate a removable electrophoresis cell accordingto an embodiment.

FIG. 13 illustrates a removable electrophoresis cell according to anembodiment.

FIG. 14A illustrates a scWB apparatus with an open lid showing theremovable electrophoresis cell according to an embodiment.

FIGS. 14B and 14C illustrate an exterior view of the scWB apparatus ofFIG. 14A showing a touchscreen feature.

FIG. 14D illustrates a scWB removable chip configured for use with thescWB apparatus of FIG. 14A.

FIGS. 15A-15C illustrate fluorescence measurements relative to proteinseparation of beta-tubulin in CHO cells performing a scWB assay via, forexample, the scWB apparatus of FIGS. 14A-14D.

FIGS. 16A-16C illustrate molecular weight sizing on 8% T gel, 10% T gel,and 12% T gel, respectively.

DETAILED DESCRIPTION

While various embodiments of the present disclosure have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions may occur to those skilled in theart without departing from the present disclosure. It should beunderstood that various alternatives to the embodiments of the presentdisclosure described herein may be employed.

As used in this specification, the singular forms “a,” “an” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, the term “a member” is intended to mean a singlemember or a combination of members, “a material” is intended to mean oneor more materials, or a combination thereof.

As used herein, the term “protein” refers to proteins, oligopeptides,peptides, and analogs, including proteins containing non-naturallyoccurring amino acids and amino acid analogs, and peptidomimeticstructures.

As used herein, the term “analyte” refers to any molecule or compound tobe detected, as described herein. Suitable analytes can include but arenot limited to, small chemical molecules such as, for example,environmental molecules, clinical molecules, chemicals, pollutants,and/or biomolecules. More specifically, such chemical molecules caninclude but are not limited to pesticides, insecticides, toxins,therapeutic and/or abused drugs, hormones, antibiotics, antibodies,organic materials, proteins (e.g., enzymes, immunoglobulins, and/orglycoproteins), nucleic acids (e.g., DNA and/or RNA), lipids, lectins,carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenicbacteria and/or eukaryotic cells such as mammalian tumor cells),viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors,nucleotides, polynucleotides, transition state analogs, inhibitors,nutrients, electrolytes, growth factors and other biomolecules and/ornon-biomolecules, as well as fragments and combinations thereof. Someanalytes described herein can be proteins such as enzymes, drugs, cells,antibodies, antigens, cellular membrane antigens, and/or receptors ortheir ligands (e.g., neural receptors or their ligands, hormonalreceptors or their ligands, nutrient receptors or their ligands, and/orcell surface receptors or their ligands).

As used herein, the term “sample” refers to a composition that containsan analyte or analytes to be detected. A sample can be heterogeneous,containing a variety of components (e.g., different proteins) orhomogenous, containing one component. In some instances, a sample can benaturally occurring, a biological material, and/or a man-made material.Furthermore, a sample can be in a native or denatured form. In someinstances, a sample can be a single cell (or contents of a single cell)or multiple cells (or contents of multiple cells), a blood sample, atissue sample, a skin sample, a urine sample, a water sample, and/or asoil sample. In some instances, a sample can be from a living organism,such as a eukaryote, prokaryote, mammal, human, yeast, and/or bacteriumor the sample can be from a virus. In some instances, a sample can beone or more stem cells (e.g., any cell that has the ability to dividefor indefinite periods of time and to give rise to specialized cells).Suitable examples of stem cells can include but are not limited toembryonic stem cells (e.g., human embryonic stem cells (hES)), andnon-embryonic stems cells (e.g., mesenchymal, hematopoietic, inducedpluripotent stem cells (iPS cells), or adult stem cells (MSC)).

The term “level,” as used herein, generally refers to variation from aplane that is orthogonal to the gravitational acceleration vector.

The present disclosure is directed to automated systems for performinganalyte detection, such as western blotting. Systems provided herein canbe used to detect analytes in samples of substantially low volume, suchas single cell analysis. In some examples, such analysis can beperformed using single cell western blotting (scWB), which employsmicrofluidic design to introduce tried-and-true microarray formats thatcombine: (i) protein molecular weight determination via electrophoresiswith (ii) protein identity determination via subsequent antibody-basedprobing of resolved protein bands. This two-stage assay can includewestern blotting. This two-stage analysis can offer ultra-highspecificity performance at the bench, without the need for costlyinfrastructure or core facilities. The scWB brings high-specificityprotein assays to single-cells, while taking advantage of the vastreagent infrastructure already available for conventional westernblotting.

Widespread implementation of scWB can impact biomedicine through: a)elucidating intratumor cell-to-cell heterogeneity, thus advancingtherapeutic efficacy of cancer drugs; b) assessing patient-specificcell-level response for companion diagnostics to targeted therapy drugcocktails; c) quantifying the purity and safety of cell-based therapiesbefore implantation, which may accelerate regulatory approval and leadto the creation of safer, more effective cell-based therapies; d)enabling analysis of rare cell populations from patients (e.g.,circulating tumor cells, CTCs) for targeted therapies; and e) enablingcharacterization of rare cell therapies such as iPSC-derived somaticcells to accelerate creation of functional tissues and in vitro diseasemodels for drug development. Single-cell western-based protein-levelinformation may provide a transformative leap towards realizingpersonalized and regenerative medicine.

To achieve microarray-like density of single-cell resolution westernblots, the scWB can use a microscope slide coated with a thinphotoactive polymer stippled with thousands of microwells having adiameter that is about 100 microns or less. In some embodiments, eachmicrowell can have a diameter in a range of about 15 microns to about 45microns in diameter (see e.g., FIG. 1). The chip can be fabricated usingsoft lithography. The chip can be removable, consumable, disposable, orcombinations thereof. The chip can be a scWB chip. The scWB workflow caninitiate when a cell suspension is dispensed on top of the microwellarray having a gel, with single cells settling into individualmicrowells passively under the action of gravity. Microwell occupancycan be controlled by limiting dilution and microwell design (e.g.,microwells sized to accommodate single cells). After cell lysis andapplication of an electric field across the gel, the photo-active gel,which can comprise benzophenone-methacrylamide, can act as a sievingmatrix that separates molecules (such as proteins) according to theirmolecular weight over short separation distances (<1 mm). With a briefUV exposure, the benzophenone-methacrylamide co-monomer can covalentlyimmobilize proteins via hydrogen abstraction, which can yield captureefficiencies approaching 100%. Thus, protein bands can be fixed inposition in the gel after the size-based separation and subsequently thegel can be probed with antibodies which can obviate the transfer andblocking steps typically used in traditional western blottingapproaches. In some instances, a scWB can be performed without pumps,valves, precise alignment or complex electrical or pneumatic interfacestypically found in scWB systems (e.g., mass spectrometers, masscytometry, flow cytometers, etc.). Multiplexing of targets can beachieved by size separation, multispectral detection, and/or repeatedstripping and re-probing of the captured proteins.

On one microscope slide, thousands or more of single cells can beassayed in parallel and as little as about 1-10 femtograms or less oftarget protein per cell can be detected which, in some instances, canimpart higher throughput and analytical sensitivity than massspectrometry. Given the widespread use of conventional western blottingand less strict antibody specificity requirements, about 10 times morecommercially-available antibodies can be validated for western blottingas compared to flow cytometry. Cell lysis can allow for measurement ofdiverse targets such as intracellular proteins, protein complexes, andpost-translational modifications that may not be accessible using flowcytometry.

Flow cytometry is an established single-cell proteomic tool with highthroughput. Yet flow cytometry can require high antibody specificity andintracellular proteins can be more challenging to detect. Mass cytometrycan make single-cell proteomic measurements but may use custom heavymetal-tagged antibodies, expensive instrumentation (e.g., $600,000 perinstrument) and high antibody specificity. Mass spectrometry is emergingas a single-cell analysis tool, yet it may require expert spectrainterpretation, and can have low throughput and low analyticalsensitivity. Automated western blotting instrumentation exists but isunable to reach single-cell resolution. In some instances, this isattributable in part to capture efficiencies of about 0.01% versus about100% for the scWB.

In some embodiments, a system for scWB can include instrumentation thatcan integrate the electrophoresis and UV capture steps. The presentdisclosure described here also provides for safety interlocking (toprevent user exposure to hazardous high voltage and UV light) andenables integrated electrophoresis and UV exposure on a microscope slideformat. While the initial application of the instrument can perform thescWB assay, the same present disclosure can enable related assays suchas small volume (but not single-cell) western blotting or small volume,parallel electrophoresis assays.

The present disclosure provides a system for performing electrophoreticseparations and photocapture of separated molecules within a thin gelattached to a planar substrate. The system can perform single-cellresolution western blotting (scWB) in a thin (<100 micron thick) gellayer on a microscope slide. In some embodiments, the system can performassays as described in, for example, PCT Patent Publication No.WO2014/138475 entitled, “Electrophoretic Separation Devices and Methodsfor Using the Same,” filed Mar. 6, 2014, the disclosure of which isincorporated herein by reference in its entirety. The present disclosurecan be a system comprising an instrument, software, associatedaccessories, and assay methods.

The system is shown in FIG. 2. The main unit 100 (also referred toherein as “instrument” and/or “device”) can be comprised of a base 101,a lid 102, an integrated computer (such as a tablet computer with atouch screen, 110), a high voltage power supply (that can be containedin a compartment 105) and a source of activation energy (e.g., radiationenergy) such as an ultraviolet (UV) light source 108. The main unit 100is configured to receive an electrophoresis cell 120 and can be used toperform an assay on a biological sample included in and/or otherwisecontained by the electrophoresis cell 120, as described in furtherdetail herein. The system can be controlled by a computer,microprocessor or microcontroller which can either be integrated intothe main unit 100 or separate from but in communication with the maininstrument body or housing (e.g., through a wired or wireless dataconnection). Integration of a computer with the instrument body canallow for flexible integration of instrument control and data managementin a compact form. For example, the tablet computer 110 can have abuilt-in sensor (e.g., camera) that can be used to read barcodes printedon a chip, for example, a scWB consumable chip. The chip can be adapted,configured, or otherwise suitable for use in scWB. The sensor can beused to read or identify one or more identification members, such asidentification numbers, on the removable electrophoresis cell. Each ofthe one or more identification members can be unique. The one or moreidentification members can include one-dimensional or two-dimensionalidentification barcodes. The one or more identification members caninclude radio frequency identification (RFID) units. The sensor canstore the one or more identification members, such as identificationnumbers, in memory coupled to the computer processor. The memory can bepart of the system or a remote computer system, such as a remote serverin network communication with the system (e.g., the “cloud”). A firstidentification member of the one or more identification members candetermine a first set of assay parameters for the assaying. The computerprocessor can be programmed to upload the first unique identificationmember, the first set of assay parameters, or a combination thereof to acomputer network. In some cases, the instrument can include detection ofthe presence of one or more target analytes and the computer processorcan be programed to upload the presence of one or more target analytes.The barcode can then be used to determine a set of assay parameters tobe used for running the barcoded chip. Once the run is complete, thetablet can upload run information along with the barcode to a computernetwork, such as a network server. The server can later be accessed by aseparate reader instrument so that imaging data and electrophoresis rundata can be associated with the same chip through the barcode.

The main unit 100 can include a frame 109 which can position and holdthe touch screen or tablet computer 110 and can hide any cableconnections to the tablet computer or screen. The source of activationenergy, such as the UV light source 108, can be a fluorescent bulb, LED,an Hg—Xe source, or other common sources. The instrument body caninclude a curved surface 112 to accommodate a cylindrical-shaped UVsource (such as a fluorescent lamp) while maintaining a compact andaesthetically pleasing profile. The exterior face of the UV light source108 can be an optical filter or a window that can reduce the lightintensity (e.g., a neutral density filter) or that can control thewavelengths of light that pass through such that optimal wavelengths canreach the scWB consumable chip. The filter or window can be heated ortreated with a coating to reduce condensation of buffer duringelectrophoresis.

In some embodiments, sensitive and high voltage components can be sealedwithin a separate compartment 105 so that any liquid (e.g., buffersolution) spilled inside the instrument 100 cannot come in contact withthe electronics. In such an example, air cooling and venting can occurfrom the bottom of the instrument. The lid 102 can rotate open aboutaxis 103 and can be supported by a friction hinge 104 which can allowthe lid 102 to stay open at various angles. The lid 102 can close ifopened less than a threshold angle (e.g., 10 degrees or any othersuitable angle). The lid 102 may include dampening mechanisms to slowthe lid 102 closure, and can latch closed using one of various methodsknown to one skilled in the art (e.g., magnetic or mechanical latches).Leveling feet 111 can be used to level the instrument base 101 such thatliquid (e.g., buffer solution) in the electrophoresis cell 120 can belevel. Leveling can be accomplished by placing a standard bubble levelin the electrophoresis cell 120 while adjusting the leveling feet 111 orthe instrument 100 can include a permanently mounted bubble level or anelectronic leveler that can interface to the computer 110 which in turncan inform the user if the instrument 100 is level. In some embodiments,the instrument 100 can include any suitable device and/or mechanismconfigured to automatically or at least semi-automatically level theinstrument.

In an example, a chip 140 (FIG. 5B), such as a disposable or consumablechip that is 75 mm long, being 1 degree “off level” can have a fluidheight that can vary by 1.3 mm across the top of the chip. If thenominal height of the fluid is only 3 mm, then being off level canresult in about a 40% variation in fluid height which can result in a40% variation in the electric field.

Interlock switches 107 can ensure that the lid 102 is closed beforehazardous UV and high voltage (HV) are enabled. The instrument 100 canbe configured to disconnect the HV power supply from the pogo pins 114when the lid 102 is opened and/or the HV supply can be disabled and theresidual charge can be dissipated to ground. The main body of theinstrument 100 may include vents which can prevent accumulation ofexplosive electrolysis gases inside the instrument body. Light blockingfeatures (such as a raised rim around the base 101 of the instrument100) may be included, which can minimize any leakage of UV light whenthe lid 102 is closed.

As shown in FIG. 3, the main instrument body or housing can include areceptacle 106 that can hold an electrophoresis cell 120. In someinstances, the electrophoresis cell 120 can be modular, removable, or acombination thereof. In an example, an electrophoresis cell, such as themodular removable electrophoresis cell 120 can be aligned using one ormore alignment pins 113 which can interface to corresponding slotsand/or holes on the modular removable electrophoresis cell 120. Thismodular design can allow for any number of removable electrophoresiscell designs to be used in the main instrument unit 100. Some suchdesigns can accommodate different chips which can fit into a recess area121 of the electrophoresis cell 120, different electrode configurations,or electrical resistance that can simulate a filled removableelectrophoresis cell to allow for convenient dry testing of the system.In some embodiments, any number of slots can be formed in the maininstrument body or housing to accommodate the one or more pins. Anelectrical connection can be formed when one or more pins are adjacentto the one or more conductive pads of the removable electrophoresis cell120. In an example, the slots can be configured to accommodate variousremovable electrophoresis cell form factors and/or various electrodeconfigurations. In an example, the slots can remain spatially fixed andthe pin configuration can be modular. In another example, the slots andpin configuration of the housing can remain spatially fixed and theconfiguration of the contact pads of the modular removableelectrophoresis cell can also remain spatially fixed. In this example,the electrical connections and mechanical features within the modularelectrophoresis cell can be reconfigured for different functions, suchas a plurality of removable electrophoresis cell designs.

Electrical connection can be made to the removable electrophoresis cell120 via spring-loaded pins, i.e., “pogo pins,” 114 which can connect toconductive contact pads 122 on the electrophoresis cell 120. Thisinterface can allow for dry contact to the HV pogo pins 114. Theelectrical contact pads 122 can be connected to conductors (for example,platinum or carbon conductors) which can contact the liquid (e.g.,buffer solution) in the removable electrophoresis cell 120. Theinstrument 100 can control the voltage difference between the pogo pins114 such that the electric field strength in the removableelectrophoresis cell can be programmatically controlled. In an example,the instrument 100 can programmatically change the polarity of the pogopin voltages such that the direction of the electric field within theremovable electrophoresis cell can also be controlled via software.Additional pins and contact pads may be added to accommodate differentremovable electrophoresis cell designs, if more than two voltages areneeded. Although not shown, in some embodiments, the receptacle 106 issurrounded by a grounding ring such that any fluid that spills outsideof the electrophoresis cell 120 during operation will be electricallygrounded once it crosses over the grounding ring. The grounding ring canbe any suitable shape, size, and/or configuration and can be formed fromany suitable grounding material.

The receptacle 106 and removable electrophoresis cells can betransparent such that an additional UV light source can be includedbelow the receptacle 106. The transparent surfaces can include lenselements to help focus light from a UV source. The imaginginstrumentation (e.g., lenses, cameras, photodetectors, etc.) can beincluded below the receptacle 106. The imaging instrumentation canenable brightfield or fluorescence imaging of the chip, before, during,or after electrophoresis. In an example, the surface of the receptacle106 can be reflective such that UV light from the UV source 108 can bereflected back to the chip to increase the UV flux. In another example,the surface of the receptacle 106 can include ultrasonic transducers (orthe like) which can transmit energy to a chip seated in the recess area121 and can assist in lysis of cells contained within the chip. In afurther example, the receptacle 106 can incorporate temperature control(heaters and/or coolers) to assist with lysis, to control heating duringelectrophoresis, or to provide temperature cycling or control fordesired chemical reactions (e.g., for melting or annealing of nucleicacid-tagged detection probes).

FIG. 4 shows an example of an electrophoresis cell, such as theremovable electrophoresis cell 120. The removable electrophoresis cell120 can have a body 120A that defines the recess area 121 designed toaccommodate a chip 140, an example of which can be a scWB consumablechip having outer dimensions equal to a standard microscope slide.Conductive contact pads 122 are coupled to the body 120A and can providean electrical interface to the instrument. Platinum wire 127 can connectfrom the contact pads 122 into the body 120A of the removableelectrophoresis cell 120. The platinum wire 127 can be inserted intononconductive tubing such that the portion of the wire 127 outside ofthe removable electrophoresis cell 120 can be electrically insulatedwhile at least of portion of the tubing can be removed so that theplatinum wire 127 inside of the removable electrophoresis cell 120 canbe exposed to facilitate contact with the buffer. To maximize activationenergy flux onto the scWB consumable chip 140, the removableelectrophoresis cell 120 can be shallow to minimize the distance betweenthe top of the slide and the source of activation energy, such as a UVsource. The removable electrophoresis cell 120 can incorporate featuresto avoid wicking of the electrophoresis buffer outside of a shallowremovable electrophoresis cell 120. For example, internal corners 123may be radiused to reduce wicking. In an example, the removableelectrophoresis cell 120 can include pouring or spout features to aid inemptying the removable electrophoresis cell when the assay is complete.

FIGS. 5A and 5B show a cross-sectional view of an electrophoresis cell,such as a modular removable electrophoresis cell 120. The chip 140 cansit flush inside of the recess area 121 such that the thin gel layer onthe top surface of the chip 140 can be level with the bottom of theremovable electrophoresis cell 120. In an example, the chip 140 can sitflush within the recess area 121 to minimize or eliminate changes inheight that can cause electric field distortions or perturbations nearthe edge of the chip 140. Electric field distortions or perturbationscan cause variations in the migration distance and in the direction ofprotein bands that are separated in the chip 140. Electrical fielddistortions can be spatial distortions or perturbations, such asdistortions near the edge of the chip 140. Electrical field distortionscan be temporal distortions or perturbations, such as bubbles passingover the chip 140. Electrical current can be proportional to a fluidheight 131 and the fluid can absorb some of the UV light from the UVlight source 108. In some instances, the fluid or liquid height (e.g.,buffer solution height) can be minimized. Selection of theelectrophoresis cell dimensions can minimize liquid height. The fluidheight 131 may be less than about 50 millimeters (mm), 20 mm, 30 mm, 10mm, 8 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm. In some instances, adistance 132 between the chip 140 and the top of the removableelectrophoresis cell 120 can be reduced to increase the flux of UV lightfrom the UV light source 108 onto the chip 140. The electrophoresis celldimensions may be selected so as to reduce the fluid height 131 andincrease the flux or power of UV light.

The distance 132 may be less than or equal to about 25 mm, 20 mm, 15 mm,14 mm, 13 mm, 12 mm, 11 mm, 10 mm, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm. Insome instances, the distance 132 is between about 1 mm and 50 mm, or 5mm and 20 mm.

The removable electrophoresis cell 120 can be designed so that the depthof the electrophoresis buffer 130 is deeper near the electrodes 128 suchthat the electrodes can be covered by the electrophoresis buffer whileallowing for a smaller fluid height 131 above the chip 140. In someinstances, the electric field strength and migration distances can varyacross the chip 140 unless the liquid (e.g., buffer solution) height 131is uniform across the chip 140. In the example shown in FIG. 5B, auniform liquid height 131 can be achieved by leveling the removableelectrophoresis cell 120, e.g., using a bubble level or electronic levelwhile adjusting the instrument feet 111, while the fluid height 131 canbe controlled by the volume of electrophoresis buffer 130 that is addedto the removable electrophoresis cell 120. Leveling features can beattached to the modular removable electrophoresis cell 120 or beintegrated into the receptacle 106 such that the removableelectrophoresis cell 120 can be leveled independent of the maininstrument body or housing 100.

In an example, the removable electrophoresis cell 120 can be configuredsuch that the lid 102 defines the fluid height 131 by creating a fixeddistance between the lid 102 and surface of the chip 140. Excess liquid(e.g., buffer solution) can be displaced such that the fluid height 131is no longer a function of the volume of electrophoresis buffer 130 orwhether or not the removable electrophoresis cell 120 is level. Thefluid height 131 can be controlled by spillover features so that oncethe target fluid level is reached, excess buffer can flow over thespillover feature into a reservoir such that the excess buffer additiondoes not change the fluid height 131.

The modular removable electrophoresis cell 120 can incorporate one ormore weir structures 124. These weir structures 124 can preventelectrolysis bubbles, which can be generated at the electrodes 128, fromdrifting over the chip 140. Bubbles above the chip 140 can distort orperturb the electric field and may block activation energy (e.g., UVlight) from the source of activation energy (e.g., UV light source) 108.When the electrophoresis buffer is poured into the removableelectrophoresis cell in the region above the electrodes 128, the weirstructures 124 can prevent bubbles already present in the buffer fromflowing across the chip 140. The weir structures 124 can help dampenfluid motion after pouring such that the buffer 130 can become quiescentmore rapidly than in a removable electrophoresis cell 120 without one ormore weir structures. In some instances, the dampening of fluid motioncan be important to reduce loss of proteins from the microwells duringlysis. The removable electrophoresis cell 120 can further incorporate asieve or one or more other features that filter out bubbles alreadypresent in the electrophoresis buffer or generated by electrolysis.

The modular removable electrophoresis cell 120 can comprise one or morestructures (126A, 126B) to capture and restrain the electrode wire 127.A hole 125 in the wall of the removable electrophoresis cell 120 canallow the electrode wire 127 to pass through the wall and to connectelectrically to the contact pads 122. The wire 127 can be sheathed in anonconductive tube (such as polytetrafluoroethylene (PTFE)). The wire127 can be sheathed in a nonconductive tube at the point where it passesthrough the hole 125 such that the wire 127 can be electricallyinsulated outside of the cell 120.

In some instances, when loading the chip 140, such as a disposable chip,into the modular removable electrophoresis cell 120, air can be trappedunder the chip 140. Air may remain trapped when the electrophoresisbuffer is added to the removable electrophoresis cell 120 and can resultin the chip 140 floating up or bubbles escaping along the edges of thechip 140 during electrophoresis, which can distort or perturb theelectric field. A droplet of buffer (e.g., 50-300 microliters) can beadded to the recess area 121. In some instances, a droplet of buffer canbe added to one side of the recess area 121 or can be added prior toloading the chip 140 into the recess area 121 after which the chip 140can be lowered into the recess area 121, allowing the liquid drop towick underneath the chip 140 to exclude any trapped air. The surfacetension of the liquid (e.g., buffer solution) under the slide (e.g.,chip) can hold the slide in the recess area 121 until theelectrophoresis buffer can be added to the cell 120. The recess area 121can be opaque or dark color which can increase the visual contrastbetween the air and liquid under the chip 140.

FIGS. 6A-6C show how a dual-purpose electrophoresis/lysis buffer can beadded to an electrophoresis cell, such as the modular removableelectrophoresis cell 120. In some examples, the dual-purpose buffer 130can be added manually wherein the user can pour the buffer 130 into themodular removable electrophoresis cell 120 in the region above theelectrode 128, can close the instrument lid 102, and can initiate theassay through the integrated computer 110. Cell lysis can occur when thedual-purpose buffer 130 is poured into the removable electrophoresiscell 120. The user can initiate the run after adding the dual-purposebuffer 130.

The instrument 100 can include a reservoir 135 which can contain thedual-purpose buffer 130 (FIG. 6A). The reservoir can have an initiallyclosed valve 136 which can prevent the buffer 130 from exiting thereservoir. The software can programmatically control the release of thebuffer 130 by opening a valve 136 allowing the buffer 130 to drain intothe modular removable electrophoresis cell 120 (FIG. 6B). The computerprocessor may separately introduce a first solution and a secondsolution from the source of solution to the chip. The first and secondsolutions may be different, such as a lysis buffer and anelectrophoresis buffer. Alternatively, the first and second solutionsmay be the same, such as washing buffers.

The computer processor may be programmed to separately introduce two ormore sequential solutions from the source of solution to the chip. Thetwo or more sequential solutions may be the same. As an alternative, thetwo or more sequential solutions may be different. In some instances,one or more solutions are manually introduced to the chip. As analternative, one or more solutions are manually introduced and one ormore solutions are programmatically introduced when the computerprocessor opens the reservoir valve 136.

Programmatic control of buffer release can provide consistent timing ofthe lysis step. The buffer 130 can be released rapidly and can fill theremovable electrophoresis cell 120 in less than about 1 second. In someinstances, the buffer 130 can fill the removable electrophoresis cell120 in less than about 0.5 seconds. In some instances, the buffer 130can fill the removable electrophoresis cell 120 in less than about 0.1seconds. The instrument 100 can include one or more active pumps. Theinstrument 100 can utilize gravity release to fill the reservoir 135with buffer 130. The reservoir 135 can be configured to contain a singlealiquot of buffer 130. In some instances, the entire volume of a singlealiquot of buffer 130 can be released when the valve 136 is opened. Insome instances, the reservoir 135 can contain larger volumes of buffer130, and a subvolume of the larger volume can be released for one assay.The release of a subvolume of buffer 130 can be controlled by theinstrument 100. In some instances, the instrument 100 can comprisemultiple reservoirs and/or the buffer 130 can be introduced into theremovable electrophoresis cell 120 at multiple locations.

Many lysis buffers can contain high concentrations of surfactants andsalts. These additives can assist in cell lysis. However, such additivescan also increase the conductivity of the buffer making such buffersless suitable for electrophoresis as excessive electrical current canlead to joule heating and loss of separation resolution. Thedual-purpose electrophoresis/lysis buffer 130 can therefore strike abalance between low conductivity for electrophoresis and the ability tolyse cells. In some instances, separately-optimized electrophoresis andlysis buffers can be used. For example, FIGS. 7A-7C show a scWB assayusing a separate electrophoresis buffer 133 and lysis buffer 134.Approximately 1 mL of lysis buffer 134 can be added directly to the topsurface of a chip, such as the disposable chip 140, to initiate lysis(FIG. 7A). After a lysis time (such as between about 5 to about 15seconds), a volume (such as about 15 mL) of electrophoresis buffer 133can be released from a reservoir 135, as in FIG. 7B. The electrophoresisbuffer 133 can wash over the surface of the chip 140 and can displacethe lysis buffer 134 and can wash it to the side of the removableelectrophoresis cell 120 (FIG. 7C) where it can be diluted by the largervolume of electrophoresis buffer 133. This dilution can significantlymitigate the impact of the higher conductivity lysis buffer 134 duringelectrophoresis. In addition, the electrical resistance of the removableelectrophoresis cell 120 can be largely determined by the thin layer offluid directly above the chip 140 (defining the liquid height 131),therefore removing the lysis buffer 134 from the region above the chip140 can greatly reduce the current during electrophoresis, independentof effect of dilution. A holding area below the electrode 128 can becreated such that the lysis buffer 134 washes into the holding regionthereby removing it from the electrical circuit entirely.

The system can also include tools and fixtures for conducting a scWBassay. FIGS. 8A-8C depict a device and method for performing antibodyprobing using a chip, which may be the scWB chip 140. The chip 140 maybe consumable. In some cases, the chip 140 is disposable or reusable. Toensure uniform antibody staining and facilitate the recovery and reuseof primary antibodies, the device can comprise an incubation chamber,such as an antibody probing fixture 150. The probing fixture 150 cancontain a rectangular region with a defined depth or recession 151 witha length and width slightly smaller than the chip 140, as in FIG. 8A.During the antibody probing step, the gel layer 142 containingimmobilized proteins (see FIG. 1) can be exposed to a uniformconcentration of detection antibodies, such as a) labeled primaryantibodies or b) unlabeled primary antibodies followed by incubationwith labeled secondary antibodies. A non-uniform gap size 152 may createa non-uniform concentration of antibody across the chip 140 during theincubation. The antibody fixtures can ensure a uniform gap that can befilled with antibody solution 138. The fixture 150 can be constructed ofa hydrophobic material (such as PTFE) or can incorporate a hydrophobiccoating such that the antibody solution cannot spread out fromunderneath the more hydrophilic gel layer 142. Keeping the antibodysolution 138 contained under the chip 140 can reduce evaporation of theantibody solution 138.

Loading of the fixture is shown in FIG. 8B. A small volume of antibodysolution 138 can be placed on the surface of the fixture. In someinstances, the small volume can be less than about 100 microliters. Thechip 140 with gel layer 142 facing down, can then be rotated onto thefixture 150 as shown in FIG. 8B. Once the chip 140 has been lowered ontothe fixture 150, the gap between the gel layer 142 and the bottom of therectangular depression (recession) 151 can be filled with antibodysolution 138. In some instances, the gap size 152 can be about 50microns or less. Once in place, the scWB consumable chip 140 can beallowed to incubate with the antibody solution 138 (see e.g., FIG. 8C).In some instances, the incubation is for about 30 to about 120 minutes.Extended incubation periods can be possible. Incubations may occur atreduced temperatures. During this incubation period, the entire fixture150 may be covered to reduce evaporative losses. A small portion of wetcloth or paper may also be placed adjacent to the fixture 150 so as tofurther limit evaporative losses. At the end of the incubation step, thechip 140 can be removed from the fixture 150 by rotating the chip 140away from the rectangular depression 151. In addition to helping tocontain the antibody solution 138 under the chip 140 during incubation,a hydrophobic fixture can facilitate recovery of the used antibodysolution 138 as the solution can tend to reform into a contiguousdroplet when the chip 140 is rotated away from the fixture 150. Primaryantibody solutions 138 may therefore be reused several times to reducecost.

The fixture 150 shown in FIGS. 8A-8C may be constructed using manycommon techniques such as machining, injection molding, or hot or coldembossing. FIG. 9 depicts a cross-sectional view of one construction foran antibody incubation fixture 155. In this example construction, arectangular gap 157 can be formed on both sides of the fixture 155 usinglaminated layers. The core 156 of the fixture 155 may be constructed ofany suitable, flat material such as for example, 0.125 inch thickpoly(methyl methacrylate) (PMMA). Thin hydrophobic layers 158, such as0.001 inch thick PTFE layers are then attached to the core layer 156.Finally, an additional hydrophobic layer having a rectangular region 159removed can be attached to the first hydrophobic layer 158. Theadvantage of using laminated layers to define the gap size is that thelaminated layers can be very flat over distances comparable to the sizeof the chip. The gap size of such a laminated device can be very uniformand can be defined by the thickness of the layer. The layers may beattached to each other using many techniques known to one skilled in theart, such as thermal bonding, solvent bonding, or use of intermediatepressure-sensitive adhesive layers.

As described with reference to FIG. 1, the first step of the scWB assaycan be to settle cells into the array of the microwells formed into thegel layer 142. Once the cells are settled into wells, the user cangently rinse the chip 140 to remove cells that have not settled intowells substantially without washing cells out of microwells. FIG. 10depicts another fixture 160 included with the system that can aid inrinsing of the chip 140 after cell settling. The rinsing fixture 160 canplace the chip 140 at a shallow angle 161 which has a typical value ofapproximately 15 degrees. In other embodiments, the angle 161 can begreater than 15 degrees or less than 15 degrees. Wash buffer 139 can beejected slowly from a pipette tip 162. The angle 161 of the rinsingfixture 160 can allow the wash buffer 139 to flow across the gel layer142 at a controlled velocity thereby minimizing perturbation of cellsthat are trapped inside microwells while rinsing away cells that remainon the surface of the gel layer 142.

The present disclosure also provides methods for obtaining molecularweight sizing of endogenous proteins contained in the cells. Separatelyoptimized lysis and electrophoresis buffers can allow for the convenientintroduction of protein standards. Preferential partitioning of proteinsto open wells versus gel can allow for facile loading of protein markersthat separate with the expected linear log molecular weight (MW) vs.mobility relationship. Thus, it may be not necessary to load distinctsub-nanoliter volumes of protein marker solutions into the thousands ofmicrowells contained within the chip. Protein markers can befluorescently tagged using a distinct wavelength such that they do notinterfere with later detection of endogenous proteins. The proteinmarkers can be tagged with a cleavable or quenchable fluorophore whichcan be detected and then removed or quenched prior to detection ofendogenous proteins. Using the two-buffer method described withreference to FIGS. 7A-7C, one can add protein markers to the lysisbuffer such that they can be introduced simultaneously as cell lysisoccurs. Alternately, protein markers can be loaded into lysosomes oragarose (or other polymer) beads. The lysosomes and beads can then besettled simultaneously along with the cells resulting in some microwellscontaining marker proteins. Proteins can leave the lysosomes or beadswhen an external electric field is supplied to commence electrophoresis.An AC electric field can be applied to release the protein markers in amanner similar to electroporation of cells. The lysis buffer can bemodified to release the protein markers from the lysosomes or beads.

Protein sizing standards can be spotted onto a chip at various locationsduring manufacturing and rehydrated during use of the chip. Separateregions on a chip, such as alternating separate regions, may bedesignated for protein sizing ladders where the wells for the sizingladders can be increased in at least one dimension to allow for moreconvenient loading of sizing markers.

Another aspect of the present disclosure allows for the introduction offluorescence standards such that detected endogenous proteins can becompared to a known fluorescence signal and measurements can morereadily be compared between chips. In an example, the fluorescencestandard can be introduced into the chip at discreet locations duringmanufacturing. Standards can be, for example, incorporated throughoutthe gel, or can be spotted onto regions of the chip before cross-linkingof the gel has completed, or the reactive groups within the gel (e.g.,benzophenone) may be activated to covalently bind the fluorescencestandards. Fluorescence standards can be introduced in beads whichco-settle into the wells along with cells. The beads can remain in thewells and the fluorescence signal from these beads can be compared tothe signal from labeled, endogenous proteins that separate away from thewells. The beads can be commercially available silica beads (or thelike), or they may be beads formed from a hydrogel. An example of ahydrogel bead includes polyacrylamide beads containing abenzophenone-methacrylamide co-monomer. Fluorescent dyes can beincorporated into the polyacrylamide bead by incubation with a desireddye followed by photo-initiated binding of the dye to benzophenonefunctional group incorporated into the hydrogel bead. Polyacrylamidebeads can allow a user to conveniently add a desired marker to thebeads. Beads can also incorporate magnetic nanoparticles to assist inmanipulation of the beads.

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 11 shows acomputer system 170 that is programmed or otherwise configured toimplement systems and methods provided herein. The computer system 170can regulate various aspects of analyte detection of the presentdisclosure, such as, for example, automated single cell westernblogging. The computer system 170 can be an electronic device of a useror a computer system that is remotely located with respect to theelectronic device. The electronic device can be a mobile electronicdevice.

The computer system 170 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 171, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 170 also includes memory or memorylocation 172 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 173 (e.g., hard disk), communicationinterface 174 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 175, such as cache, other memory,data storage and/or electronic display adapters. The memory 172, storageunit 173, interface 174, and peripheral devices 175 are in communicationwith the CPU 171 through a communication bus (solid lines), such as amotherboard. The storage unit 173 can be a data storage unit (or datarepository) for storing data. The computer system 170 can be operativelycoupled to a computer network (“network”) 176 with the aid of thecommunication interface 174 via a wired or wireless connection. Thenetwork 176 can be the Internet, an internet and/or extranet, or anintranet and/or extranet that is in communication with the Internet. Thenetwork 176 in some cases is a telecommunication and/or data network.The network 176 can include one or more computer servers, which canenable distributed computing, such as cloud computing. The network 176,in some cases with the aid of the computer system 170, can implement apeer-to-peer network, which may enable devices coupled to the computersystem 170 to behave as a client or a server.

The CPU 171 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 172. The instructionscan be directed to the CPU 171, which can subsequently program orotherwise configure the CPU 171 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 171 can includefetch, decode, execute, and writeback, and/or any other suitableoperation.

The CPU 171 can be part of a circuit, such as an integrated circuit. Oneor more other components of the computer system 170 can be included inthe circuit. In some cases, the circuit is an application specificintegrated circuit (ASIC). In some embodiments, the CPU 171 and/orintegrated circuit can include and/or can execute one or more modulesassociated with controlling the systems described herein. As used hereinthe term “module” refers to any assembly and/or set ofoperatively-coupled electrical components that can include, for example,a memory, a processor, electrical traces, optical connectors, software(executing in hardware), and/or the like. For example, a module executedin the processor can be any combination of hardware-based module (e.g.,a field-programmable gate array (FPGA), an ASIC, a digital signalprocessor (DSP)) and/or software-based module (e.g., a module ofcomputer code stored in memory and/or executed at the processor) capableof performing one or more specific functions associated with thatmodule.

The storage unit 173 can store files, such as drivers, libraries,profiles, saved programs, etc. The storage unit 173 can store user data,e.g., user preferences and user programs. The computer system 170 insome cases can include one or more additional data storage units thatare external to the computer system 170, such as located on a remoteserver that is in communication with the computer system 170 through anintranet or the Internet (e.g., network attached storage (NAS) device).

The computer system 170 can communicate with one or more remote computersystems through the network 176. For instance, the computer system 170can communicate with a remote computer system of a user (e.g., serviceprovider). Examples of remote computer systems include servers, hostdevices, personal computers (e.g., portable PC), slate or tablet PC's(e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones(e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personaldigital assistants. The user can access the computer system 170 via thenetwork 176.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 170, such as, for example, on the memory172 or electronic storage unit 173. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 171. In some cases, the code canbe retrieved from the storage unit 173 and stored on the memory 172 forready access by the processor 171. In some situations, the electronicstorage unit 173 can be precluded, and machine-executable instructionsare stored on memory 172.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 170, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks, or the like, also may be considered as media bearing thesoftware. As used herein, unless restricted to non-transitory, tangible“storage” media, terms such as computer or machine “readable medium”refer to any medium that participates in providing instructions to aprocessor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium, or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables, copper wires, and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a compact disc-readonly memory (CD-ROM), digital video disc (DVD) or digital videodisc-read only memory DVD-ROM, any other optical medium, punch cardspaper tape, any other physical storage medium with patterns of holes, arandom-access memory (RAM), a read-only memory (ROM), a programmableread-only memory (PROM) and erasable programmable read-only memory(EPROM), a FLASH-EPROM, any other memory chip or cartridge, a carrierwave transporting data or instructions, cables or links transportingsuch a carrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 170 can include or be in communication with anelectronic display 177 that comprises a user interface (UI) 178 forproviding, for example, an output or readout of the system coupled tothe computer processor. Examples of UI's include, without limitation, agraphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 171. Thealgorithm can, for example, be used to analyze results, such as thepresence of one or more target analytes in a biological sample. A finaldata set can be constructed based on portions of data gathered from eachassay of each biological sample.

While the removable electrophoresis cell 120 is particularly describedabove with reference to FIGS. 2-9, in other embodiment, a scWBinstrument such as the instrument 100 can be configured to receiveand/or otherwise use any suitable electrophoresis cell. For example,FIGS. 12A and 12B illustrate a removable electrophoresis cell 220according to an embodiment. In some embodiments, the removableelectrophoresis cell 220 has been designed and/or otherwise optimizedfor manufacturing by injection molding. Portions of the removableelectrophoresis cell 220 can be similar in form and/or function tocorresponding portions and/or features of the electrophoresis cell 120described above with reference to FIGS. 2-9 and thus, such portions arenot described in further detail herein.

As shown, the removable electrophoresis cell 220 includes wicking breaks229 to prevent fluid from wicking up the electrodes to one or morecontact pad areas 222. The electrophoresis cell 220 also includes anintegrated chip lifter 265 that facilitates removal of a chip (e.g., thechip 140) from the cell 220. In the embodiment shown in FIGS. 12A and12B, when the chip lifter 265 is in the down position, it fits flushwith the electrophoresis body so as to prevent trapping of air bubblesas well as electric field perturbations that can be caused by changes influid height near the edge of the chip (as described in detail abovewith reference to the electrophoresis cell 120). The electrophoresiscell 220 includes weirs 224 that are inserted separately, allowing forthe electrophoresis cell 220 to be molded. To attach the electrode wire(not shown), heat staking features 267 are included. The wire can bestretched between the heat staking features 267 which are then melted topermanently hold the wire in place. A small (<3 mm) circular recess 266allows an alternate way of removing the chip from the electrophoresiscell 220 by using a pointed prying tool. The small size of the recess266 minimizes bubble trapping and electric field perturbations. As shownin FIG. 12B, the underside of the electrophoresis cell 220 includesstabilization posts 269 which force the electrophoresis cell 220 tocontact the receptacle in an instrument (e.g., the instrument 100) atfixed points. As shown, the electrophoresis cell 220 includes spoutfeatures 268 that can assist in pouring used buffer from theelectrophoresis cell after use. In this manner, the electrophoresis cell220 can be used in any suitable scWB process and/or procedure such asany of those described herein.

EXAMPLES

An integrated opto-electronic instrument has been built that accuratelycontrols all timed steps and provides uniform, repeatable, safeapplication of electric field and UV illumination for execution of thescWB assay. The integrated instrument minimizes the time betweenseparation and UV photocapture, thus limiting diffusive dispersion andloss of separation resolution. For safe deployment to external users,mechanical interlocking of the high voltage and UV light has beenimplemented. Below, details on the optimized system components anddemonstrate safe operation and acceptable assay performance of theintegrated prototype are presented.

Removable electrophoresis cell. Iterative prototyping and designprocesses were employed to build, test, and optimize a removableelectrophoresis cell for use in the final integrated scWB device. Thedesign of the removable electrophoresis cell was configured to, forexample, reduce electrolysis bubble formation near the electrodes thatspread to cover the liquid surface within the cell, perturbing theelectric field and blocking a portion of the UV light during thesubsequent UV exposure step. Further, the design was configured to, forexample, increase reliable chip adhesion and repeatable buffer addition.

In some embodiments, a chip recess can include finger cut-outs forfacile chip removal. Such a chip recess can be sufficiently deep toprotect the chip from being dislodged during buffer addition obviatingthe need for a petroleum jelly fixative. Further, bubble trapping weirstructures can be used to separate the platinum electrodes from the maincell compartment and prevent bubbles from floating over the chipsurface. In some instances, however, such finger cut-outs can lead to,for example, electric field distortion observed at the edges of the chip(8° near edge vs. <2° near center). Numerical simulations confirm that,in some instances, this non-uniformity can be caused by liquid (e.g.,buffer solution) height discontinuities due to finger cutouts and thechip not being flush with the bottom surface of the cell. Changes in theliquid height at the edge of the chip can cause the electric field linesto diverge, leading to electric field distortion near the edges of thechip.

To increase field uniformity, changes in the liquid height were avoided.For example, in some embodiments, a removable electrophoresis cell 320includes a chip recess 321 configured to fully recess the chip such thatthe gel layer protruded beyond the chip recess 321 and finger cut-outswere removed (see FIG. 13). Such a removable electrophoresis cell 320can incorporate the following designs: 1) A relatively large bufferreservoir allowing for the addition of lysis/electrophoresis bufferwithout spilling, 2) bubble traps (e.g., weirs 324) to preventelectrolysis bubbles from migrating over the surface of the chip andblocking UV light during photocapture while further damping fluid motionabove the chip, 3) pogo-pin high-voltage contacts for integration withthe instrument lid, and 4) the chip recess 321 without finger cutouts tominimize field distortion near the edges of the chip. The removableelectrophoresis cell 300 can also include electrical contact pads 322and a platinum electrode 327, as described above.

Light source selection. Several candidate light sources were tested.Benzophenone capture chemistries are known to be activated in the rangeof 330-360 nanometers (nm) and the Lightning cure lamp has a broadspectrum output with significant power at 316 nm and 365 nm. Severalcompact light sources in the 310 to 365 nm wavelength range wereevaluated. No capture was observed under high power (1200 microWatts(mW)/cm²) 365 nm LEDs (Hamamatsu).

Compact UV fluorescent tube lamps were obtained from UV Systems (Renton,Wash.) and the performance of bulbs was compared with broad outputscentered at 365 nm and 312 nm, respectively. Both bulbs have significantpower output in the 330-360 nm range. The 312 nm wavelength bulb yieldeda significant increase in capture efficiency compared to a 365 nm bulb(69% vs. 10% for a 4 minute exposure). Reducing the exposure time on the312 nm bulb to 1 min yielded a measured mean capture efficiency of 52%(compared to 69% for a 4 min exposure time) suggesting that the exposuretime can be reduced from 4 minutes with a moderate reduction in thecapture efficiency.

Integrated Prototype. An integrated high-voltage electrophoresis and UVexposure unit is used to accurately control all timed steps and provideuniform, repeatable, safe application of electric fields and UVillumination. An integrated opto-electronic instrument 300 was builtthat incorporates the custom-designed removable electrophoresis cell320, a chip 340 (such as those described above), and a 312 nm UV bulbfrom UV Systems to enable safe and automated sequential operation ofelectrophoresis and UV exposure steps (see e.g., FIGS. 14A-14D). Atouchscreen 310 with a graphical user interface was developed to allowusers to have control over key assay steps: lysis time, electrophoresisrun time, voltage, and UV exposure time (FIGS. 14B and 14C).

Safety interlocking features were successfully incorporated and the UVlight was visually observed to cease upon opening of the lid. Theelectrical current was measured with a multimeter and found to ceaseupon opening the lid. The instrument was additionally fitted with adrain circuit to drain any residual charge from the high voltage pinwhen the lid is opened. Light-blocking features were incorporated tolimit any leakage of UV light to negligible levels.

Methods and Analysis: The ability to lyse cells settled in a scWB chipby covering the chip with ˜1 mL of lysis buffer for 10-15 seconds andthen quickly replacing the lysis buffer with electrophoresis buffer wasdemonstrated. Loss of lysed proteins inside the microwells can beminimized by rapidly replacing the buffer above the chip withoutexcessive fluid perturbation using the removable electrophoresis celland instrumentation described with reference to FIG. 4, FIGS. 5A and 5B,and FIGS. 7A-7C.

Preferential partitioning of proteins to open wells versus gel allowsfor facile loading of protein markers that separate with the expectedlinear log MW vs. mobility relationship (see FIGS. 16A-16C).

Once incubated with the chip, the marker-containing buffer was removedto avoid continuous injection and reduced resolution of the proteinmarkers during separation. Using the two-buffer workflow (see, e.g.,FIGS. 7A-7C), the ability to introduce molecular weight sizing proteinmarkers in the lysis buffer has been shown. Surface tension holds themarker-containing lysis buffer on the surface of the gel, filling thewells, until the electrophoresis buffer is introduced and flows over thechip. Separate from the need to avoid continuous injection, introductionof the protein markers in the smaller volume of lysis buffer (versus theelectrophoresis buffer) reduces the required mass (and cost) of ladderproteins by an order of magnitude. Preliminary results for sizing ofendogenous beta-tubulin in a Chinese hamster ovary (CHO) cell line usingovalbumin and immunoglobulin G (IgG) markers reduced variability fromfield perturbations across the chip (raw migration distance had a 7.9%CV while a sizing correction using ovalbumin and IgG reduced the CV to3.4%) (see e.g., FIGS. 15A-15C, illustrating a relationship between anamount of measured fluorescence and an amount of protein separation).FIGS. 16A-16C illustrate molecular weight sizing on 8% T gel, 10% T gel,and 12% T gel, respectively.

As described above, some embodiments herein relate to a computer storageproduct with a non-transitory computer-readable medium (also can bereferred to as a non-transitory processor-readable medium) havinginstructions or computer code thereon for performing variouscomputer-implemented operations. The computer-readable medium (orprocessor-readable medium) is non-transitory in the sense that it doesnot include transitory propagating signals per se (e.g., a propagatingelectromagnetic wave carrying information on a transmission medium suchas space or a cable). The media and computer code (also can be referredto as code) may be those designed and constructed for the specificpurpose or purposes. Examples of non-transitory computer-readable mediainclude, but are not limited to, magnetic storage media such as harddisks, floppy disks, and magnetic tape; optical storage media such asCDs, DVDs, CD-ROMs, and holographic devices; magneto-optical storagemedia such as optical disks; carrier wave signal processing modules; andhardware devices that are specially configured to store and executeprogram code, such as ASICs, Programmable Logic Devices (PLDs),Read-Only Memory (ROM), and Random-Access Memory (RAM) devices. Otherembodiments described herein relate to a computer program product, whichcan include, for example, the instructions and/or computer codediscussed herein.

Some embodiments and/or methods described herein can be performed bysoftware (executed on hardware), hardware, or a combination thereof.Hardware modules may include, for example, a general-purpose processor,a field programmable gate array (FPGA), and/or an ASIC. Software modules(executed on hardware) can be expressed in a variety of softwarelanguages (e.g., computer code), including C, C++, Java™, Ruby, VisualBasic™, and/or other object-oriented, procedural, or other programminglanguage and development tools. Examples of computer code include, butare not limited to, micro-code or micro-instructions, machineinstructions, such as produced by a compiler, code used to produce a webservice, and files containing higher-level instructions that areexecuted by a computer using an interpreter. For example, embodimentsmay be implemented using imperative programming languages (e.g., C,FORTRAN, etc.), functional programming languages (Haskell, Erlang,etc.), logical programming languages (e.g., Prolog), object-orientedprogramming languages (e.g., Java, C++, etc.) or other suitableprogramming languages and/or development tools. Additional examples ofcomputer code include, but are not limited to, control signals,encrypted code, and compressed code.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations, or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

Where methods and/or events described above indicate certain eventsand/or procedures occurring in certain order, the ordering of certainevents and/or procedures may be modified. Additionally, certain eventsand/or procedures may be performed concurrently in a parallel processwhen possible, as well as performed sequentially as described above.

1. A system for assaying a biological sample for a presence of a targetanalyte, the system comprising: an assaying device having a housing, theassaying device comprising: a receptacle disposed in the housing andconfigured to accept an electrophoresis cell, the electrophoresis cellhaving a recess area configured to accept a chip including a polymericseparation medium with activatable functional groups that covalentlybond to the target analyte when activated, the chip configured to acceptthe biological sample; and a source of activation energy configured tosupply activation energy to activate the activatable functional groups;and a computer controller operably coupled to the source of activationenergy, the computer controller operable to activate the source ofactivation energy to direct an application of activation energy to thepolymeric separation medium to activate the activatable functionalgroups. 2.-3. (canceled)
 4. The system of claim 1, wherein the computercontroller is included in an electronic device remotely situated withrespect to the housing and is in communication with the assaying devicevia at least one of a wired and wireless network.
 5. (canceled)
 6. Thesystem of claim 1, wherein the source of activation energy is a sourceof ultraviolet radiation.
 7. (canceled)
 8. The system of claim 1,wherein the assaying device includes a power source disposed in thehousing, the power source being electrically coupled to at least oneelectrode on opposing sides of the recess area of the electrophoresiscell when the electrophoresis cell is disposed in the receptacle, thepower source being configured to provide power to the at least oneelectrode to generate an electric field across the recess area.
 9. Thesystem of claim 8, wherein the computer controller is configured tocontrol an electric field strength of the electric field across time.10. The system of claim 1, wherein the assaying device includes aleveling sensor configured to sense a degree of leveling of a solutionin the recess area and a leveling indicator configured to provide anindication of the degree of leveling of the solution in the recess area,a relatively high degree of leveling results in a relatively low degreeof spatial perturbations in the electric field.
 11. (canceled)
 12. Thesystem of claim 1, wherein the computer controller is operably coupledto a sensor configured to identify an identification member on the chipwhen the electrophoresis cell is inserted into the receptacle, theidentification member determining a set of assay parameters for theassaying. 13.-20. (canceled)
 21. The system of claim 1, furthercomprising: a rinsing fixture, wherein an angle of the rinsing fixturecontrols a flow rate of buffer solution to the chip.
 22. The system ofclaim 1, further comprising: a probing fixture adjacent to the chip. 23.An apparatus, comprising: a housing including a base and a lid; areceptacle defined by an interior surface of the base, the receptacleconfigured to receive an electrophoresis cell, the electrophoresis cellconfigured to be coupled to a chip including a polymeric separationmedium with functional groups configured to covalently bond to a targetanalyte within a biological sample disposed on the chip in response tobeing activated; a radiation energy source coupled to an interiorsurface of the lid, the radiation energy source configured to supplyactivation energy operable to activate the functional groups; anelectric power source disposed within the housing and fluidicallyisolated from the receptacle, the electric power source configured to beelectrically coupled to the electrophoresis cell when theelectrophoresis cell is disposed in the receptacle; and a computercontroller coupled to the housing and fluidically isolated from thereceptacle, the computer controller operable to activate the radiationenergy source and the electric energy source when the electrophoresiscell is disposed in the receptacle to assay the biological sample.24.-26. (canceled)
 27. The apparatus of claim 23, wherein the radiationenergy source is electromagnetic radiation from at least one of visiblelight, ultraviolet (UV) light, or infrared light.
 28. The apparatus ofclaim 23, wherein an external face of the radiation energy sourceincludes an optical filter configured to pass a subset of wavelengthscompatible with the assaying through the optical filter, the subset ofwavelengths being within an UV spectrum of wavelengths. 29.-31.(canceled)
 32. The apparatus of claim 23, wherein the housing defines aplurality of slots, each slot from the plurality of slots configured toaccommodate a pin electrically connected to the electric power source,wherein an electrical connection is formed when at least one pin isadjacent to a conductive contact pad of the electrophoresis cell, theconductive contact pad being electrically connected to the at least oneelectrode.
 33. The apparatus of claim 23, wherein the computercontroller is configured to direct a voltage polarity of at least onepin to control a direction of the electric field. 34.-35. (canceled) 36.The apparatus of claim 23, wherein the housing is coupled to a pluralityof leveling feet, the leveling feet configured to be adjusted relativeto the housing to level a solution relative to the chip. 37.-38.(canceled)
 39. The apparatus of claim 23, wherein the housing includes asource of solution, the computer controller is configured to direct flowof a solution from the source of solution to the chip when theelectrophoresis cell is within the receptacle.
 40. The apparatus ofclaim 39, wherein the source of solution is operatively coupled to avalve, the computer controller operable to transition the valve betweenan open configuration and a closed configuration to direct a flow ofsolution from the source of solution to the chip when theelectrophoresis cell is within the receptacle.
 41. The apparatus ofclaim 39, wherein the source of solution contains at least a firstsolution and a second solution, the computer controller is configured toseparately introduce at least the first solution and the second solutionfrom the source of solution to the chip when the electrophoresis cell iswithin the receptacle. 42.-61. (canceled)
 62. A system for use in anassay of a biological sample, the system comprising: an electrophoresiscell having a recess area and at least one electrode; a chip configuredto receive the biological sample, the chip being disposable in therecess area such that a surface of a polymeric separation medium of thechip and a non-recessed surface of the electrophoresis cell aresubstantially flush; and an assaying device having: a housing, areceptacle disposed in the housing and configured to accept theelectrophoresis cell, a source of activation energy configured to supplyactivation energy to the polymeric separation medium when theelectrophoresis cell is in the receptacle, and an electric power sourcedisposed within the housing and fluidically isolated from thereceptacle, the electric power source configured to provide electricpower to the at least one electrode to generate an electric field acrossthe recess area when the electrophoresis cell is in the receptacle, thesubstantially flush surfaces operable to reduce distortion in theelectric field.
 63. The system of claim 62, wherein the source ofactivation energy is a source of ultraviolet radiation.
 64. The systemof claim 62, wherein the assaying device includes a leveling sensorconfigured to sense a degree of leveling of a solution in the recessarea, wherein a relatively high degree of leveling results in arelatively low degree of spatial perturbations in the electric field.65. The system of claim 62, further comprising: a rinsing fixture,wherein an angle of the rinsing fixture controls a flow rate of buffersolution to the chip.
 66. The system of claim 62, wherein the assayingdevice further includes a source of solution configured to provide aflow of a solution from the source of solution to the chip when theelectrophoresis cell is within the receptacle.
 67. The system of claim62, further comprising: a conductive contact pad coupled to a body ofthe electrophoresis cell and electrically connected to the at least oneelectrode, the conductive contact pad configured to be electricallyconnected to the electric power source of the assaying device when theelectrophoresis cell is disposed within the receptacle to electricallyconnect the electric power source to the at least one electrode.
 68. Thesystem of claim 62, wherein the electrophoresis cell includes a weirstructure disposed on at least one side of the recess area, a bottomsurface of the weir structure being above the non-recessed surface ofthe electrophoresis cell, the weir structure configured to control aflow of a solution below the bottom surface of the weir structure andacross the recess area.
 69. The system of claim 68, wherein the weirstructure is configured to prevent bubbles from floating above a topsurface of the chip when the electrophoresis cell is within thereceptacle.